Stabilized Collagen and Elastin-Based Scaffolds for Mitral Valve Tissue Engineering.

There is a significant clinical need for new approaches to treatment of mitral valve disease. The aim of this study was to develop a tissue-engineered mitral valve scaffold possessing appropriate composition and structure to ensure ideal characteristics of mitral valves, such as large orifice, rapid opening and closure, maintenance of mitral annulus-papillary muscle continuity, in vivo biocompatibility and extended durability. An extracellular matrix-based scaffold was generated, based on the native porcine mitral valve as starting material and a technique for porcine cell removal without causing damage to the matrix components. To stabilize these structures and slow down their degradation, acellular scaffolds were treated with penta-galloyl glucose (PGG), a well-characterized polyphenol with high affinity for collagen and elastin. Biaxial mechanical testing presented similar characteristics for the PGG-treated scaffolds compared to fresh tissues. The extracellular matrix components, crucial for maintaining the valve shape and function, were well preserved in leaflets, and in chordae, as shown by their resistance to collagenase and elastin. When extracted with strong detergents, the PGG-treated scaffolds released a reduced amount of soluble matrix peptides, compared to untreated scaffolds; this correlated with diminished activation of fibroblasts seeded on scaffolds treated with PGG. Cell-seeded scaffolds conditioned for 5 weeks in a valve bioreactor showed good cell viability. Finally, rat subdermal implantation studies showed that PGG-treated mitral valve scaffolds were biocompatible, nonimmunogenic, noninflammatory, and noncalcifying. In conclusion, a biocompatible mitral valve scaffold was developed, which preserved the biochemical composition and structural integrity of the valve, essential for its highly dynamic mechanical demands, and its biologic durability.

Functional Heart Valve Scaffolds Obtained by Complete Decellularization of Porcine Aortic Roots in a Novel Differential Pressure Gradient Perfusion System.

There is a great need for living valve replacements for patients of all ages. Such constructs could be built by tissue engineering, with perspective of the unique structure and biology of the aortic root. The aortic valve root is composed of several different tissues, and careful structural and functional consideration has to be given to each segment and component. Previous work has shown that immersion techniques are inadequate for whole-root decellularization, with the aortic wall segment being particularly resistant to decellularization. The aim of this study was to develop a differential pressure gradient perfusion system capable of being rigorous enough to decellularize the aortic root wall while gentle enough to preserve the integrity of the cusps. Fresh porcine aortic roots have been subjected to various regimens of perfusion decellularization using detergents and enzymes and results compared to immersion decellularized roots. Success criteria for evaluation of each root segment (cusp, muscle, sinus, wall) for decellularization completeness, tissue integrity, and valve functionality were defined using complementary methods of cell analysis (histology with nuclear and matrix stains and DNA analysis), biomechanics (biaxial and bending tests), and physiologic heart valve bioreactor testing (with advanced image analysis of open-close cycles and geometric orifice area measurement). Fully acellular porcine roots treated with the optimized method exhibited preserved macroscopic structures and microscopic matrix components, which translated into conserved anisotropic mechanical properties, including bending and excellent valve functionality when tested in aortic flow and pressure conditions. This study highlighted the importance of (1) adapting decellularization methods to specific target tissues, (2) combining several methods of cell analysis compared to relying solely on histology, (3) developing relevant valve-specific mechanical tests, and (4) in vitro testing of valve functionality.

Design and Testing of a Pulsatile Conditioning System for Dynamic Endothelialization of Polyphenol-Stabilized Tissue Engineered Heart Valves.

Heart valve tissue engineering requires biocompatible and hemocompatible scaffolds that undergo remodeling and repopulation, but that also withstand harsh mechanical forces immediately following implantation. We hypothesized that reversibly stabilized acellular porcine valves, seeded with endothelial cells and conditioned in pulsatile bioreactors would pave the way for next generations of tissue engineered heart valves (TEHVs). A novel valve conditioning system was first designed, manufactured and tested to adequately assess TEHVs. The bioreactor created proper closing and opening of valves and allowed for multiple mounting methods in sterile conditions. Porcine aortic heart valve roots were decellularized by chemical extractions and treated with penta-galloyl glucose (PGG) for stabilization. Properties of the novel scaffolds were evaluated by testing resistance to collagenase and elastase, biaxial mechanical analysis, and thermal denaturation profiles. Porcine aortic endothelial cells were seeded onto the leaflets and whole aortic roots were mounted within the dynamic pulsatile heart valve bioreactor system under physiologic pulmonary valve pressures and analyzed after 17 days for cell viability, morphology, and metabolic activity. Our tissue preparation methods effectively removed cells, including the potent α-Gal antigen, while leaving a well preserved extra-cellular matrix scaffold with adequate mechanical properties. PGG enhanced stabilization of extracellular matrix components but also showed the ability to be reversible. Engineered valve scaffolds encouraged attachment and survival of endothelial cells for extended periods and showed signs of widespread cell coverage after conditioning. Our novel approach shows promise toward development of sturdy and durable TEHVs capable of remodeling and cellular repopulation.